The present invention relates to an etidoproteolytic hatching enzyme denoted zonase. This novel enzyme is a non-selfdegrading endoprotease. A procedure for extracting said enzyme in waste water from hatcheries producing Atlantic salmon larvae, and a simple procedure for obtaining up to sequence-grade purity of this special endoprotease, which turns out to possess rather unique proteolytic characteristics is described.
Proteases in purified states are increasingly used in research, in laboratory and clinical analysis, and in food production procedures. Demand is increasing, especially for enzymes with properties commensurate with specific applications. This has stimulated quests for new sources of proteases, which allow safe, sustainable and economical modes of production.
Here we exploit a new, rich source of endoproteases connected to aquaculture of Atlantic salmon. An essential aspect of this industry is the hatchery-production of developing eggs, which hatch to yield larva. Hatching is accomplished by embryos producing endoproteases, which, when secreted, effectively and specifically split the eggshell open to allow the larva to swim out and start life on its own (Refs.: Yamagami 1988; Walther 1993).
The crucial enzymes causing fish hatching have only been characterized in a few fishes, and in almost all cases, these hatching enzymes have been interpreted to be metallo-proteases (Refs.: Hagenmaier 1974: Ohzu and Kasuya 1979; Schoots and Denucxc3xa8 1981; DiMichele et al. 1981; Yasumasu et al. 1989 a, b. Araki and Onozato 1990; Hung et al. 1997). Genetic structure of some hatching enzymes has only recently become available (Ref.: Yasumasu et al. 1992).
Putative hatching enzymes have also been reported in invertebrates, where again most such enzymes have been interpreted as being metalloproteases (e.g. Barrett and Edward 1976; Lepage and Gache 1989; Roe and Lennarz 1990). However, a few strong cases for serine protease-like zonases have been reported (e.g. Post et al 1988). Conversely, among higher vertebrates than fish, putative hatching enzymes have also been reported.
For instance, both Urch and Hedrick (1981; concerning amphibians) and Yamazaki et al (1994; concerning mouse) reported zonases, which appeared to be serine proteases. The biological and biochemical rationale behind two different types of hatching enzymes among hatching animals is at present not fully understood.
Non-selfdegrading endoproteases are unique. In nature the destructive effects of proteases on biological tissues mandate that it is a functional adaption that proteases should self-destruct. For instance, this is the case for pancreatic endoproteases which otherwise might damage and ulcerate the intestine with serious injury to the individual. The situation is dramatically different during the hatching process for fish. If the crucial enzymes allowing hatching by degrading the eggshell (chorion, zona, or vitteline envelope) were to attack each other by proteolysis, the eggshell may survive intact and the individual would not be born, but die intra ovo. Hence the enzyme must selectively target and cleave the eggshell, and not other entities of enzyme molecules. Even enzymatic affinity for the fish larva itself should be avoided since this will damage the larva and expose it to serious risk of microbial and other diseases. Hence there is a straightforward biological rationale for why zonase should be non-selfdegrading.
Enzymatic activity similar to the zonase hatching enzyme of Atlantic salmon has been observed in eggs of several marine fish species. Since the proposal of Walther (1993; 2000) is that degrading of the zona by a zonase is a fundamental and general aspect of sexual reproduction, the implication is that zonase-like enzymes may be found in a wide variety of fishes and in other vertebrates, where they have so far gone undetected due to the need to follow special isolation procedures during purification.
The zonase according to the present invention is present in large amounts in the waste waters and hatching fluids of salmon eggs. In this crude aqueous state, salmon zonases may be effectively readied for purification by conventional techniques. This source of zonases offers a great advantage compared to isolation of enzymes from whole embryos, since it effectively obviates complications from extraneous biomaterial (eggshells, embryos and larvae). Thus, enzyme purification becomes greatly simplified.
An additional advantage is that the developmentally-staged salmon eggs may be transferred to minimal volumes of water prior to hatching. When highly synchronous hatching is induced by elevated (room) temperatures, or by deoxygenaton (Oppen-Berntsen et al. 1990), this yields a small volume of highly concentrated preparation of crude zonases.
A further essential aspect of the method is that, despite the increasing concentration of the proteolytic zonase, the stability of its resident zonase was observed to remain intact. Furthermore, it is important to note that this procedure yields zonase enzyme in a medium of almost pure water, containing at most 1 mM NaCl, but where zonases nevertheless possess and retain full enzymatic integrity over time. This preparation is therefore a valuable starting material for subsequent preparations of proteolytic zonases in various degree of purification, up to sequence-grade purity.
Zonase according to the present invention is a novel hatching enzyme of the serine-endoprotease family. This new enzyme has been shown to be non-selfdegrading. The inventors have purified the zonase to sequence grade, at which point it consist of only two closely similar enzymatic moieties. This was made possible due to the described method, which set forth the sequence in which the purification method has to be performed. The said zonase catalyses sequence specific endoproteolysis of peptide bonds C-terminal to basic amino acid residues, said specific endoproteolysis follows after the amino acids lysine or arginine. The zonase is further stable against inactivation conditions such as 4-8M urea or more, molar salt concentrations, distilled water and organic solvents such as at least 33% dioxane. It is resistant to auto inactivation both as dry substance and in aqueous solutions and retains its enzymatic activity in solution at about 15-30xc2x0 C. preferably about 22xc2x0 C. until 30-70 days preferably about 50 days, and in a solution at about 0xc2x0 C. for more than six months. The zonase is inactivated in 10% (v/v) of xcex2-mercaptoethanol and reactivated by removal of xcex2-mercaptoethanol by evaporation by a temperature up to about 50xc2x0 C.
The claimed zonase is a valuable tool for many well-known processes presently employing endoproteases. These include among many;
analytical work generating specific peptides from proteins (see Example 6),
addition of endoproteases to laundry detergents to enhance the action of detergents and allow laundry washing at low temperatures,
enhancing availability of costly ingredients for brewery processes,
cleaning bakery utensils with little or no use of detergents to obtain bio-usable materials instead of waste,
enhancing bioavailability of nutrients in feed by pretreating feed with proteases or using a neutral and stable zonase protease as a type of gastric digestive support in young fish and other newborn vertebrates.
The zonase from salmon is distinct from well known hatching enzymes from medaka (xe2x80x9cchorionasexe2x80x9d). By cloning and sequencing in salmon a gene homologous to the chorionase gene of the medaka, it was found that the enzyme resulting from transcription and translation of this gene did not crossreact at all with a highly specific polyclonal antibody to salmon zonase. Salmon zonase is thus a novel enzyme compared to known hatching enzymes.